In recent years, genomic-based assay techniques have uncovered many potential drug targets. This advance has lead to interest and development in the field of proteomics in general, and different kinds of screening assays in particular. Since genes encode for proteins, and proteins, in turn, perform nearly all of the life functions in a cell, then, virtually nothing is more important than deciphering the functions of proteins, because proteins are the targets against which most drugs are designed to act upon. Of the various approaches that have been proposed, array-based expression analysis and mutation mapping of many genes have made a major impact on biology and on drug discovery and development. Given that it is costly to sort out the multitude of chemical or drug targets one by one, this fact has created a demand for screening technologies that enable robust and parallel analysis of many targets.
Together with genomics, advanced chemical technologies and high-throughput screening, protein microarray technology has the potential to aid in understanding biological systems or system biology, as well as in developing new medicines of the future. Functional protein microarrays use native proteins as probes arranged on a substrate surface. Arrays of this type are useful for parallel studies of the activities of native proteins, such as protein-protein and protein-small molecule interactions. The interaction of proteins with a surface, however, complicates the preparation of protein microarrays. This problem arises because (i) proteins can denature at the interface between an aqueous solution and a solid surface, and (ii) random immobilization of proteins on a surface may cause the active site(s) of the proteins to be inaccessible for binding of targets. To achieve maximum binding capacity and desired stability of proteins on a surface with largely preserved structure and activity, the surface of solid supports generally need to be re-engineered. Examples include “deformable” polymer-grafted surfaces for immobilization of proteins (e.g., HydrogGel coated slides, PerkinElmer Life Science, Boston, Mass.), amine- or thiol-reactive surfaces for covalent coupling of proteins, or functional group-presenting surfaces for specific binding of proteins. Functional group-presenting surfaces include avidin-coated surfaces for biotinylated proteins, Ni+2-chelating surfaces for histidine-tagged proteins, or antibody-modified surfaces for native proteins.
Traditionally, biological microarrays have been fabricated or printed on substrate surfaces that are largely two-dimensional, such as those of glass slides. Recently, porous substrates for biological microarrays have been proposed and reduced to practice. (See e.g., International Patent Applications No. WO0116376 A1, or WO0061282 A1, incorporated herein.) Porous substrates have several advantages over conventional two-dimensional substrates. These advantages include, for example, a higher loading capacity for probes in each microspot with an associated potential higher binding capacity, and generally, a higher binding specificity for target molecules, as well as greater accessibility of targets to the probes in each microspot, which increases the likelihood that a target reacts with its complement probe. This latter phenomenon, it is believed, is a result of the three-dimensional nature of a porous surface in which a significant portion of probes are captured in the micro- or nano-channels in the porous matrix.
Conventional porous slides or other substrates typically are constructed with a contiguous porous layer. In such a situation, even though individual microspots in an array may be distinct and physically separated from each other, the underlying porous matrix is not. This physically undifferentiated construction leads to problems associated with contamination or crosstalk. When multiple samples are applied to a single substrate, the sample solutions tend to spread and merge together by means of capillary wicking through underlying, interconnected channels in the porous substrate Hence, porous substrates have not readily been used for multiplexed assays on a single substrate. This limitation deprives the multiplexed applications of a readily available resource and its associated advantages.
The present invention overcomes the crosstalk problems, thereby extending the applications of porous substrates for bioassays using immobilized biological or chemical molecules for arrays in a microplate format. For instance, the production of identical DNA and protein arrays in the wells of a standard format microplate can be of great benefit for high-throughput analysis, as each resultant microplate will allow parallel processing of many different test samples against the same or different replicate biological arrays. In combining the unique properties of porous substrates with the high throughput capability of standard format microplates, one can achieve superior performance of surface-mediated bioassays including biological microarrays.